Assessing the effects of combinations of genetic mutations in heterogenous populations of cells (bacteria, eukaryotic etc.) has implications in metabolic and genetic engineering, disease diagnostics and synthetic biology. Particularly, individual genetic mutations conferring a phenotype when found alone, may result novel or unpredictable phenotypes in the presence of other mutations. But current genotyping methods do not adequately assess the effects of combinations of mutations found in heterogenous populations and there are presently no high-resolution and high-throughput techniques for screening combinations of mutations that exist in a population.
Assembly of diverse genetic elements into a single vector traditionally required restriction and ligation enzyme-based methods that are time-consuming and laborious. For example, each sub-cloning step requires the resulting clone be screened and characterized before the introduction of additional fragments. Clones produced by blunt end ligation require confirmation that the fragment was introduced in the proper orientation, while sticky-end ligation requires that the restriction sites utilized to produce the sticky ends on the acceptor fragment also be present in the donor fragment, but not at a site that would interrupt the sequence of interest within the donor fragment. Thus, the selection of workable restriction sites depends entirely on the compositions of the pieces being joined and must be carefully considered in each case. Moreover, the efficiency of such restriction-enzyme based cloning methods is limited by the number of nucleic acid molecules that can be ligated together in a single reaction.
It has been shown that simultaneous amplification of more than one DNA segment can be achieved with a Multiplex polymerase chain reaction (PCR) using primers tagged with unrelated nucleotide sequences which are then ligated together into a single DNA molecule (Chamberlain et al. (1988) Nucleic Acids Research 16 (23): 11141-11156). But PCR products amplified with primers lacking the unrelated nucleotide sequence are not reliably produced due to differences in hybridization kinetics among the primers, and it is therefore necessary to tag each primer with an identical nucleotide sequence to achieve efficient amplification of multiple sequences. All of the PCR products then contain identical unrelated sequences which have to be removed or extended before they could be linked to form one DNA molecule containing all sequences of interest.
One method of amplifying several DNA segments which occur in non-adjacent portions of a DNA sample, termed “splicing by overlap extension” (U.S. Pat. No. 5,023,171), assembles DNA molecules at precise junctions without the use of restriction enzymes or ligase. Component fragments to be recombined are generated in separate polymerase chain reactions using uniquely designed primers which produce amplicons having complementary termini to one another. Upon mixing and denaturation of these amplicons, strands having complementary sequences at their 3′ ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a nucleic acid molecule in which the original sequences are spliced together. Subsequent rounds of PCR amplify the resulting spliced polynucleotide. This technique requires time to optimize primer sequences and amplification conditions to produce desired products. Each junction between the fragments to be spliced together must be individually considered, and a pair of primers must be designed for each target DNA fragment in order to make the ends compatible. Considerations for the design of PCR primers, (e.g., melting temperature, G-C content, avoidance of hairpin and dimer formation, and stringency for false priming sites) become increasingly complex as the number of fragments to be spliced in the reaction increases, such that combining more than just three or four target DNA segments becomes an insurmountable PCR reaction design problem. In addition, splicing by overlap extension performs the linker tagging and amplification in each site in a separate reaction, and subsequent reactions are used to assemble the pieces. This limits the usefulness of this technique from a genotyping approach although it is an effective gene construction technique.
Thus, despite advances in recombinant DNA technology, there exists a need for improved methods that provide for the rapid and ordered 1-step assembly of non-adjacent polynucleotides from a heterogenous DNA population. Particularly needed are methods which can facilitate the assembly of a number of polynucleotides with minimal manipulation and characterization of intermediate products, into a single DNA molecule in suitable quantity for accurate characterization of mutations within the assembled DNA fragments and with efficient, high throughput processing that will enable the characterization of multiple mutations that interact to create a specific phenotype. These and other needs can be met by the methods of the present invention.